GSM6504652: 293T-CUT-Tag-invivo-Hemin-TMPyP4; Homo sapiens; OTHER
Following Hemin treatment, cells were collected and lysed with Trizol reagent. Total RNA was extracted according to the manufacturer's instructions. Biotin-PEG4-Hemin was combined with the Tn5-based CUT&Tag strategy to identify Hemin binding sites in the genome. Briefly, one million HEK293T  cells were resuspended in NE1 buffer (20 mM HEPES at pH 7.9, 10 mM KCl, 0.5 mM spermidine,0.1% Triton X-100, 20% Glycerol, 1× protease inhibitors) and incubated on ice for 10 minutes. After centrifugation at 1, 300 g for 10 minutes, nuclei were isolated and collected. These nuclei were washed once with PBS and resuspended with 100 μL of wash buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors). 10 μL pre-activated concanavalin A coated magnetic beads (Smart-Lifesciences) were added to these nuclei and incubated on a rotator for 10 minutes at room temperature. The supernatant was removed and bead-bound nuclei were resuspended in 100 μL of dig-wash buffer (0.05% digitonin, 20 mM HEPES at pH 7.5,150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) containing 2 mM EDTA.Biotin-PEG4-Hemin was added to the sample and rotated overnight at 4°C in the dark.For the control sample, biotin-PEG4-hydrazide was used to replace biotin-PEG4-Hemin. These nuclei were washed three times in 200 μL of dig-wash buffer to remove unbound biotin-PEG4-Hemin or biotin-PEG4-hydrazide. Then anti-biotin rabbit mAb was diluted 100-fold in 100 μL of dig-wash buffer with 2 mM EDTA and was added to the nuclei with one-hour rotation at room temperature in the dark. After washing once with dig-wash buffer, these nuclei were resuspended with 100 μL of dig-wash buffer with 2 mM EDTA and 1 μL of mouse anti-rabbit IgG, incubated for another one hour at room temperature in the dark, and washed three times with dig-wash buffer. A 1:250 dilution of pA-Tn5 adapter complex (~0.2 μM) was prepared in 100 μL of dig-300 buffer (0.05% digitonin, 20 mM HEPES at pH 7.5, 300 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) and added to the nuclei followed by one-hour rotation at room temperature in the dark. After five washes with dig-300 buffer, the bead-bound nuclei were resuspended with 40 μL of tagmentation buffer (10 mM TAPS-NaOH at pH 8.5, 10 mM MgCl 2 , 7.5% DMF) and incubated at 37°C for one hour. To terminate the tagmentation reaction, 1.5 μL of 0.5 M EDTA, 0.5 μL of 10% SDS and 1 μL of 20 mg/mL proteinase K were added and incubated at 55°C for 1 hour and then for 15 minutes at 75°C. The DNA was extracted with Sera-Mag carboxylate-modified magnetic beads (GE Healthcare) for library preparation. 21 μL DNA was mixed with a universal i5 and a uniquely barcoded i7 primer, and amplified with NEB Q5 high- fidelity 2× master mix. The libraries were purified with 0.56-0.85× volume of Sera- Mag carboxylate-modified magnetic beads and subjected to the Bioanalyzer DNA analysis and Illumina sequencing. ChIP-Rx was performed with 1×10 7 human cells and 1×10 6 MEF cells for spike-in normalization as described in a previous study [21]. For immunoprecipitation, sonicated chromatin was incubated with 10 μg specific antibodies and 15 μL of pre- blocked Protein A/G beads (Smart-Lifesciences). After extensive washes, the captured DNA was eluted for library preparation using the NEBNext Ultra II DNA library prep kit for Illumina before sequencing on a NovaSeq 6000. G4-CUT&Tag and R-loop CUT&Tag were performed as previously described( Wang et al. 2021; Li et al. 2021)